CN116179603A - SINV vector for stably expressing exogenous gene and preparation method and application thereof - Google Patents

Abstract

The invention discloses a sindbis virus vector for stably expressing exogenous genes, a preparation method and application thereof. The SINV infectious viral particles carrying the green fluorescent protein genes are successfully prepared by using the Sindbis virus vector transfected cells. The infectious viral particles can be subjected to in vitro multi-round screening to obtain adaptive mutation, can infect various cells in vitro, can replicate and proliferate after infection, can improve the stability of exogenous inserted genes, can spread across multi-level synapses in the forward direction of a rat brain nerve loop, and has wide application value in the aspects of brain nerve loop marking and tracing, marking of non-human primate nerve cells, establishment of a drug screening platform, development of a drug inhibition virus action mechanism, development of virus vaccines and diagnostic reagents, establishment of animal models, virus replication, analysis of pathogenic mechanisms and the like.

Description

SINV vector for stably expressing exogenous gene and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a sindbis virus vector for stably expressing exogenous genes, and a preparation method and application thereof.

Background

There are a very large number of different types of neurons in the central nervous system, whose dendrites and axons together constitute a complex neural network. These complex neural networks enable the body to perform normal physiological activities such as learning, cognition, memory, fear, etc. Abnormal occurrence of the neural network may also cause corresponding neural diseases such as parkinson's disease and the like. Analysis of the cerebral nerve loop is the basis for a greater insight into brain function and neurological disease, and viral tracer tools can help to analyze the cerebral nerve loop. Thus, the development of viral tracer tools provides more options for mapping brain connections.

Sindbis virus (SINV) belongs to the family togaviridae, genus alphavirus, whose genome is a single-stranded positive strand RNA of about 12kb in length, and encodes a total of 5 structural proteins (capsid, E3, E2, 6K, and E1) and 4 non-structural proteins (NSP 1, NSP2, NSP3, and NSP 4). Sindbis virus is transmitted in nature mainly by mosquito bites in vertebrates such as birds, mammals, has a wide host range including humans, mice, monkeys, etc., and can infect the nervous system. This infectious nature can make it a vector that mediates the introduction of foreign genes into the host. In addition, the nature of sindbis virus infection of the brain nervous system provides it with the ability to resolve the nerve loops. However, the sindbis virus vector has instability of inserting exogenous genes, the inserted exogenous genes are easily sheared and lost by viruses in the continuous amplification process, and the expression of the exogenous genes is gradually reduced or even completely lost. Similar conditions exist for many RNA viruses, but the specific cleavage mechanism is currently unknown. There is a need for an infectious vector of sindbis virus capable of expressing a foreign gene more stably.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Sindbis virus infectious vector capable of more stably expressing exogenous genes after the adaptive mutation is screened by laboratory pressurization, virus particles and application thereof in cerebral neuron markers. The SINV virus vector capable of expressing the green fluorescent protein gene more stably is successfully prepared by transfecting cells with the infectious vector.

The invention provides a directed evolution method based on continuous passage and laboratory pressurized screening, which is used for screening mutant strains which are more stably expressing exogenous genes than wild type, sequencing the mutant strains to obtain 853 nucleotide sequences of NSP1 genes, mutating G into A, mutating corresponding 285 amino acid sequences from Gly into Ser, and carrying out mutation to obtain Sindbis virus infectious vectors capable of more stably expressing green fluorescent protein genes, wherein pSINV is used as a skeleton vector, and fluorescent protein genes EGFP are connected to the skeleton vector; the Sindbis virus infectious vector is sequentially connected with a UBC promoter, a 5'UTR, a nucleotide sequence of an NSP1 gene, a nucleotide sequence of an NSP2 gene, a nucleotide sequence of an NSP3 gene, a nucleotide sequence of an NSP4 gene, a nucleotide sequence of a C gene, a nucleotide sequence of an E3 gene, a nucleotide sequence of an E2 gene, a nucleotide sequence of a 6K gene, a nucleotide sequence of an E1 gene, a nucleotide sequence of an EGFP gene and a 3' UTR; the 853 nucleotide sequence of NSP1 gene is mutated from G to A, and the corresponding 285 amino acid sequence is mutated from Gly to Ser. The nucleotide sequence of the NSP1 gene mutation of the Sindbis virus infectious vector is shown as SEQ ID NO. 7.

The invention also provides sindbis virus particles which are prepared by transfecting cells with the sindbis virus vector.

The invention also provides a method for preparing the sindbis virus particle, and cells are transfected by using the sindbis virus vector.

Further, the cells are BHK-21 cells. BHK-21 cells are convenient to culture, and Sindbis virus can quickly replicate and efficiently express exogenous genes on the cells.

The invention also provides the use of the sindbis virus particles in forward cross-multistage synaptic transmission.

The invention also provides application of the Sindbis virus particles in marking of non-human primate nerve cells and nerve loop tracing.

The invention also provides application of the sindbis virus particles in research of neuroscience problems.

Further, the neuroscience problem includes, but is not limited to, the following:

analysis of the mechanism of viral action, viral replication and pathogenic mechanism of drug inhibition.

The invention also provides application of the Sindbis virus particles in preparing medicines for marking neurons. Because the sindbis virus particles of the present invention are confirmed to have the effect of labeling neurons, they can be used for the preparation of a drug for labeling neurons.

In conclusion, compared with the prior art, the invention achieves the following technical effects:

1. the invention provides a Sindbis virus system for efficiently, conveniently and more stably expressing exogenous proteins, which does not need the steps of in vitro transcription and RNA transfection, and can directly transfect cells by using sindbis virus infectious vectors, thereby saving time and operation steps. Compared with a wild sindbis vector, the virus prepared by the system can more stably express the inserted exogenous gene by the mutated sindbis vector, and is more beneficial to the research on sindbis virus infectious vector.

2. The invention has important value for developing in-vivo labeling neurons and analyzing nerve loops, and has important practical significance and wide application value for other application researches (such as protein expression, gene therapy, labeling of non-human primate nerve cells, nerve loop labeling, drug screening, epitope analysis, novel vaccine and diagnostic reagent, and the like) and basic researches (such as replication, packaging, pathogenic mechanism, and the like).

3. The prepared virus particles can mark mouse brain nerve cells, and the sindbis virus particles can be observed to spread two-stage neurons in a mouse neural network in a forward and trans-synaptic way within 96 hours.

4. Analysis of the structure of the neural loop is the basis for developing brain science research, and a good tool for marking the neural loop has important significance for analyzing the structure of the neural loop. SINV is capable of infecting nerve cells of animals such as humans and mice and has the potential to act as a marker of nerve loops. The sindbis virus particles of the invention can be used not only in non-primates, but also in brain science research in non-human primates.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the strategy for obtaining adaptive mutation Sindbis virus by pressure screening directed evolution and the construction of adaptive mutation and wild Sindbis virus infectious vector.

FIG. 2 shows the construction strategy of the adaptive mutation Sindbis virus infectious vector related vector obtained by the pressurized screening directed evolution and the plaque condition of the infectious vector after mutation.

FIG. 3 fluorescent expression profile and one-step growth curve of the adaptive mutant Sindbis virus infectious vector of the present invention after transfection.

FIG. 4 shows the expression stability of the exogenous genes of the sindbis virus particles carrying EGFP gene and wild sindbis virus particles carrying EGFP gene after the strain mutation of the present invention.

FIG. 5 prediction of NSP1 and NSP2 protein structure of the adaptive mutated Sindbis virus infectious vector of the present invention.

FIG. 6 neural loop markers of the sindbis virus particles carrying EGFP gene of the present invention with adaptive mutations on the mouse brain.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

All reagents, raw materials, technical services and instrumentation used in the present invention are commercially available, except as specifically described.

Example 1

The invention discloses a Sindbis virus infectious vector with adaptive mutation after directed evolution, which is prepared by the following steps:

the green fluorescent-expressing Sindbis virus infectious vector obtained after transfection of cells with pSINV-EGFP (Shi, xiang-Wei et al, "A new anterograde trans-sy naptic tracer based on Sindbis virus," Neural regeneration research vol.17,12 (2022): 2761-2764.Doi: 10.4103/1673-5374.339495) was serially passaged on BHK-21 cells, the green fluorescent-expressing viral plaques were picked up by multiple rounds of plaques, and fresh BHK-21 cells were re-infected, 6 rounds of screening were repeated, plaques with green fluorescent protein were picked up all the time, and the culture was expanded, and the supernatant was collected, and viral RNA extraction and sequencing analysis and alignment of the key genes were performed, the results of which are shown in FIG. 1A. Compared with the wild type, this example obtained a point mutation at 853 amino acid of NSP1 gene, and enabled the Sindbis virus vector to express the foreign gene more stably.

SINV-WT, SINV-EGFP and SINV-G285S-EGFP (structure shown in FIG. 1B) all start transcription and translation of viral structural proteins and non-structural proteins by UBC promoter, and complete the packaging of the virus. The primers shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are used for amplifying the two fragments respectively, so that the point mutation of the 853 nucleotide sequence of NSP1 is introduced.

The PCR reaction system was 50. Mu.l: 5 x Reaction Buffer:10 μl,10mM dNTPs:1 μl,10 μM Forward Primer:2.5 μl,10 μM Reverse Primer:2.5 μl, template DNA:0.5 μl, DNA Polymerase:0.5 μl, nuclear-Free Water:33 μl. The amplification conditions were: 98℃60s,98℃10s,55℃15s,72℃60s,72℃10min,16℃10min,30 cycles.

pSINV-EGFP was digested with PacI and BglII, then the fusion fragment was inserted into pSINV-EGFP using Vazyme homologous recombination kit, the recombinant product was transformed into competent HB101, clones identified as positive by PCR were cultured and plasmids were extracted for sequencing, and the clone with the correct mutation was sequenced and named pSINV-G285S-EGFP.

FIG. 1B is a schematic diagram showing the construction of the Sindbis virus infectious vector of the present example. FIG. 1B is a schematic diagram of the structure of a Sindbis virus infectious vector that does not carry the EGFP gene; "pSINV-EGFP" is a schematic structural diagram of a Sindbis virus infectious vector carrying the EGFP gene; "pSINV-G285S-EGFP" is a schematic structural diagram of an adaptive mutant Sindbis virus infectious vector carrying the EGFP gene. NSP 1-4, C, E3, E2, 6K and E1 are Sindbis virus proteins.

Example 2

The adaptive mutation Sindbis virus infectious vector related vector construction strategy and the infectious vector plaque condition after mutation:

after the plasmids pSINV-WT, pSINV-EGFP, pSINV-G285S-EGFP, pSINV-D422G-EGFP and pSINV-G285S/D422G-EGFP shown in FIG. 2A were extracted with a plasmid extraction kit, BHK-21 cells were transfected with lipofectamine 2000 at 37℃at 5% (v/v) CO ₂ After 48 hours of incubation in an incubator, the supernatants were collected and infected with BHK-21 cells, and double-layer plaque was performed to observe the plaque status of several different infectious vectors, and the results are shown in FIG. 2B. The infectious vector containing the G285S mutation has a reduced plaque phenotype change. Through infection of the present example, SINV-G285S-EGFP infectious vectors prepared sindbis virus particles having a phenotype different from that of the wild type can be obtained.

Example 3

Fluorescent expression after transfection of the adaptive mutation Sindbis virus infectious vector and one-step growth curve:

pSINV-WT, pSINV-EGFP and pSIN of example 1 were extracted with plasmid extraction kitAfter the V-G285S-EGFP plasmid, BHK-21 cells were transfected with lipofectamine 2000 at 37℃with 5% (V/V) CO ₂ The generated virus infects BHK-21 cells, the cell state and fluorescence expression are observed by an inverted fluorescence microscope at different time points, and the result is shown in figure 3A, and a green fluorescence signal can be generated after 12 hours, which indicates that the wild sindbis virus infectious vector carrying EGFP gene and the infectious vector after adaptive mutation can successfully package SINV virus and efficiently and rapidly express exogenous proteins. After a part of the virus supernatant collected at different time points is split-charged, a part of the virus supernatant collected at different time points is taken, the virus titer of the supernatant collected at different time points is detected by a double-layer plaque method, a one-step growth curve is drawn, and the result is compared with wild type viruses without EGFP genes and wild type viruses with EGFP genes, and the result is shown in figure 3B. The growth curves of the three vectors are not significantly different, which indicates that the infectious vector carrying the EGFP gene after the adaptive mutation does not influence the replication of the Sindbis virus infectious vector. The remaining supernatant was kept at-80℃for further use in subsequent experiments. Through the infection of the example, SINV-G285S-EGFP infectious vector prepared SINV-G285S-EGFP infectious vector was obtained.

Example 4

The sindbis virus particle carrying EGFP gene after the allergic mutation and the wild sindbis virus particle carrying EGFP gene are amplified in vitro by passage, and the expression stability of the exogenous gene is the same:

the viral supernatant obtained in example 2 was designated as P0 generation, each of the P0 generation viral particles was infected with fresh BHK-21 cells at 1MOI, the supernatant obtained after 24 hours was designated as P1 generation, each of the P1 generation viral particles was further infected with fresh BHK-21 cells at 1MOI, the supernatant obtained after 24 hours was designated as P2 generation, and serial subculturing was sequentially performed until P5 generation viral supernatant was obtained. The results of the fluorescent expression for each generation are shown in FIG. 4A. The SINV-EGFP fluorescent expression gradually decreases, but the SINV-G285S-EGFP fluorescent expression is not obviously decreased. RNA is extracted from virus supernatant of each generation, primers shown in SEQ ID NO.8 and SEQ ID NO.9 are adopted for carrying out one-step RT-PCR amplification, the loss condition of inserted genes is detected, and the result is shown in a figure 4B, the loss condition of wild SINV-EGFP virus begins to appear in the generation P3, and more than half of fluorescence is lost in the generation P5. Through continuous passage infection of the embodiment, SINV-G285S-EGFP infectious vector prepared sindbis virus particles having the characteristic of expressing foreign genes more stably than wild type can be obtained.

Example 5

Prediction of NSP1 and NSP2 protein structures of the adaptive mutated Sindbis virus infectious vectors of the present invention:

the sequences of NSP1 and NSP2 genes shown in example 1 before and after mutation were subjected to prediction of protein structure by alpha fold2, and the results are shown in FIG. 5. There were slight differences in protein structure before and after mutation. Through the prediction of the embodiment, the predicted structures of the NSP1 and NSP2 proteins of the Sindbis virus can be obtained, and the influence of the Missense3D predicted point mutation on the protein structure is found to possibly cause the structural change of the NSP1 protein after the point mutation, so that the virus particles have a more stable effect of exogenous gene insertion after the adaptive mutation.

Example 6

The adaptive mutant sindbis virus particle carrying EGFP gene of the invention marks the nerve loop of the cumulus on the mouse brain:

150nL of SINV-G285S-EGFP and SINV-EGFP virus particles obtained in example 2 were taken (virus titers were 2.8X10, respectively) ⁹ PFU/mL and 3.2X10 ⁹ PFU/mL) was positioned for injection into the upper hill of C57BL/6 mice (20-25 g), animals were anesthetized 96 hours after virus injection, perfused with 0.9% (V/V) saline, then fixed with 4% (V/V) paraformaldehyde, brain tissue was removed and immersed in 4% (V/V) paraformaldehyde solution, and then placed in 30% (V/V) sucrose solution for 2 days; the bottom of the brain tissue is cut flat, the brain tissue is placed on a base, embedded and frozen, then sliced, and the brain slice is taken and observed by using a fluorescence microscope. As a result, as shown in fig. 6, fig. 6A is an injection schematic and projection path of the Superior Colliculus (SC) to the lateral posterior thalamus nucleus (LP) to the Lateral Amygdala (LA); fig. 6B shows fluorescence signals observed at the injection site both on the colliculus, across the primary neurons to the lateral posterior thalamus nucleus and across the two-stage neurons to the lateral amygdala. SINV-G285S-EGFP and SINV-EGFP virus has consistent trans-synaptic properties in the mouse brain. Thus, it was shown that both the post-mutation sindbis virus and the wild-type virus can only be transmitted across the mouse brain region via cis-trans synapses.

By combining the above embodiments, the invention provides a novel adaptive mutation Sindbis virus infectious vector which is obtained by directional evolution through laboratory pressurized screening and is used for more stably expressing inserted exogenous genes, virus particles thereof and application thereof in cerebral neuron markers. The SINV virus carrying the green fluorescent protein gene is successfully prepared by transfecting cells with an infectious vector. The virus can replicate and proliferate after infection, can infect various cells in vitro, can forward cross-multistage synapse transmission in a rat brain nerve loop, and has wide application value in the aspects of brain nerve loop marking and tracing, marking of non-human primate nerve cells, establishment of a drug screening platform, drug inhibition virus action mechanism, research and development of virus vaccines and diagnostic reagents, establishment of animal models, analysis of virus replication and pathogenic mechanism and the like.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

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Claims (9)

1. The sindbis virus vector for stably expressing the exogenous gene is characterized in that the sindbis virus vector takes pSINV as a skeleton vector, and fluorescent protein gene EGFP and point mutated NSP1 gene are connected to the skeleton vector;

the sindbis virus vector is sequentially connected with a nucleotide sequence of a UBC promoter, a 5'UTR, a nucleotide sequence of an NSP1 gene, a nucleotide sequence of an NSP2 gene, a nucleotide sequence of an NSP3 gene, a nucleotide sequence of an NSP4 gene, a nucleotide sequence of a C gene, a nucleotide sequence of an E3 gene, a nucleotide sequence of an E2 gene, a nucleotide sequence of a 6K gene, a nucleotide sequence of an E1 gene, a nucleotide sequence of an EGFP gene and a 3' UTR; the nucleotide sequence of the UBC promoter is shown as SEQ ID NO. 5; the nucleotide sequence of the 5' UTR is shown as SEQ ID NO. 6; the nucleotide sequence of the NSP1 gene is shown as SEQ ID NO.7, and the 853 nucleotide sequence of the NSP1 gene is mutated from G to A; the nucleotide sequence of the NSP2 gene is shown as SEQ ID NO. 8; the nucleotide sequence of the NSP3 gene is shown as SEQ ID NO. 9; the nucleotide sequence of the NSP4 gene is shown as SEQ ID NO. 10; the nucleotide sequence of the C gene is shown as SEQ ID NO. 11; the nucleotide sequence of the E3 gene is shown as SEQ ID NO. 12; the nucleotide sequence of the E2 gene is shown as SEQ ID NO. 13; the nucleotide sequence of the 6K gene is shown as SEQ ID NO. 14; the nucleotide sequence of the E1 gene is shown as SEQ ID NO. 15; the nucleotide sequence of the fluorescent protein gene EGFP is shown as SEQ ID NO. 16; the nucleotide sequence of the 3' UTR is shown as SEQ ID NO. 17.

2. A sindbis virus particle for stably expressing a foreign gene, which is prepared by transfecting cells with the sindbis virus vector of claim 1.

3. The method for preparing sindbis virus particles stably expressing foreign genes according to claim 2, wherein cells are transfected using the sindbis virus vector according to claim 1.

4. The method of claim 3, wherein the cells are BHK-21 cells.

5. Use of sindbis virus particles according to claim 2 for forward transmission across multiple synapses.

6. Use of sindbis virus particles according to claim 2 for labeling of non-human primate nerve cells and for nerve loop tracking.

7. Use of sindbis virus particles according to claim 2 in neuroscience research.

8. The use of claim 7, wherein the neuroscience problem includes, but is not limited to, the following:

analysis of the mechanism of viral action, viral replication and pathogenic mechanism of drug inhibition.

9. Use of sindbis virus particles according to claim 2 for the preparation of a medicament for labeling neurons.

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